A preliminary study on the properties and fouling-release performance of siloxane–polyurethane coatings prepared from poly(dimethylsiloxane) (PDMS) macromers

Siloxane–polyurethane fouling-release (FR) coatings based on aminopropyl terminated poly(dimethylsiloxane) (PDMS) macromers were prepared and characterized for FR performance via laboratory biological assays. These systems rely on self-stratification, resulting in a coating with a siloxane-rich surface and polyurethane bulk. Previously, these coating systems have used PDMS with multiple functional groups which react into the polyurethane bulk. Here, aminopropyl terminated PDMS macromers were prepared, where a single amine group anchors the PDMS in the coating. Coatings were prepared with four molecular weights (1000, 5000, 10,000, and 15,000 g mol^?1) and two levels of PDMS (5% and 10%). High water contact angles and low surface energies were observed for the coatings before and after water immersion, along with low pseudobarnacle removal forces. Laboratory bioassays showed reduced biofilm retention of marine bacteria, good removal of diatoms from coatings with low molecular weight PDMS, high removal of algal sporelings (young plants), and low removal forces of live barnacles.     

A preliminary study on the properties and fouling-release performance of siloxane-polyurethane coatings prepared from poly(dimethylsiloxane) (PDMS) macromers

Siloxane-polyurethane fouling-release (FR) coatings based on aminopropyl terminated poly(dimethylsiloxane) (PDMS) macromers were prepared and characterized for FR performance via laboratory biological assays. These systems rely on self-stratification, resulting in a coating with a siloxane-rich surface and polyurethane bulk. Previously, these coating systems have used PDMS with multiple functional groups which react into the polyurethane bulk. Here, aminopropyl terminated PDMS macromers were prepared, where a single amine group anchors the PDMS in the coating. Coatings were prepared with four molecular weights (1000, 5000, 10,000, and 15,000 g mol?1) and two levels of PDMS (5% and 10%). High water contact angles and low surface energies were observed for the coatings before and after water immersion, along with low pseudobarnacle removal forces. Laboratory bioassays showed reduced biofilm retention of marine bacteria, good removal of diatoms from coatings with low molecular weight PDMS, high removal of algal sporelings (young plants), and low removal forces of live barnacles.     

Enhancing the efficacy of fouling-release coatings against fouling by Mytilus galloprovincialis using nanofillers

Fouling-release (FR) coatings minimise the adhesion strength of fouling organisms. This study describes improved technologies to control the settlement and adhesion of the important fouling organism Mytilus galloprovincialis by incorporating the nanofillers titanium dioxide (TiO₂) and carbon nanotubes (CNTs) in polydimethylsiloxane (PDMS) matrices. The incorporation of TiO₂ prevented larval settlement when photoactivated with UV light, even at the lowest concentration of the nanofiller (3.75 wt%). Notably, there was 100% mortality of pediveligers exposed to photoactivated TiO₂. However, plantigrades initially settled to photoactivated TiO₂, but their adhesion strength was significantly reduced on these surfaces in comparison to blank PDMS. In addition, plantigrades had high mortality after 6 h. In contrast to the enhanced antifouling and FR properties of PDMS incorporating TiO₂, the incorporation of CNTs had no effect on the settlement and adhesion of M. galloprovincialis.     

Combinatorial materials research applied to the development of new surface coatings IV. A high-throughput bacterial biofilm retention and retraction assay for screening fouling-release performance of coatings

Abstract

A high-throughput bacterial biofilm retention screening method has been augmented to facilitate the rapid analysis and down-selection of fouling-release coatings for identification of promising candidates. Coatings were cast in modified 24-well tissue culture plates and inoculated with the marine bacterium Cytophaga lytica for attachment and biofilm growth. Biofilms retained after rinsing with deionised water were dried at ambient laboratory conditions. During the drying process, retained biofilms retracted through a surface de-wetting phenomenon on the hydrophobic silicone surfaces. The retracted biofilms were stained with crystal violet, imaged, and analysed for percentage coverage. Two sets of experimental fouling-release coatings were analysed with the high-throughput biofilm retention and retraction assay (HTBRRA). The first set consisted of a series of model polysiloxane coatings that were systematically varied with respect to ratios of low and high MW silanol-terminated PDMS, level of cross-linker, and amount of silicone oil. The second set consisted of cross-linked PDMS-polyurethane coatings varied with respect to the MW of the PDMS and end group functionality. For the model polysiloxane coatings, HTBRRA results were compared to data obtained from field immersion testing at the Indian River Lagoon at the Florida Institute of Technology. The percentage coverage calculations of retracted biofilms correlated well to barnacle adhesion strength in the field (R² = 0.82) and accurately identified the best and poorest performing coating compositions. For the cross-linked PDMS-polyurethane coatings, the HTBRRA results were compared to combinatorial pseudobarnacle pull-off adhesion data and good agreement in performance was observed. Details of the developed assay and its implications in the rapid discovery of new fouling-release coatings are discussed.     

Combinatorial materials research applied to the development of new surface coatings IV. A high-throughput bacterial biofilm retention and retraction assay for screening fouling-release performance of coatings

A high-throughput bacterial biofilm retention screening method has been augmented to facilitate the rapid analysis and down-selection of fouling-release coatings for identification of promising candidates. Coatings were cast in modified 24-well tissue culture plates and inoculated with the marine bacterium Cytophaga lytica for attachment and biofilm growth. Biofilms retained after rinsing with deionised water were dried at ambient laboratory conditions. During the drying process, retained biofilms retracted through a surface de-wetting phenomenon on the hydrophobic silicone surfaces. The retracted biofilms were stained with crystal violet, imaged, and analysed for percentage coverage. Two sets of experimental fouling-release coatings were analysed with the high-throughput biofilm retention and retraction assay (HTBRRA). The first set consisted of a series of model polysiloxane coatings that were systematically varied with respect to ratios of low and high MW silanol-terminated PDMS, level of cross-linker, and amount of silicone oil. The second set consisted of cross-linked PDMS-polyurethane coatings varied with respect to the MW of the PDMS and end group functionality. For the model polysiloxane coatings, HTBRRA results were compared to data obtained from field immersion testing at the Indian River Lagoon at the Florida Institute of Technology. The percentage coverage calculations of retracted biofilms correlated well to barnacle adhesion strength in the field (R(2)=0.82) and accurately identified the best and poorest performing coating compositions. For the cross-linked PDMS-polyurethane coatings, the HTBRRA results were compared to combinatorial pseudobarnacle pull-off adhesion data and good agreement in performance was observed. Details of the developed assay and its implications in the rapid discovery of new fouling-release coatings are discussed.     

A comparison of the antifouling/foul-release characteristics of non-biocidal xerogel and commercial coatings toward micro- and macrofouling organisms

Five non-biocidal xerogel coatings were compared to two commercial non-biocidal coatings and a silicone standard with respect to antifouling (AF)/fouling-release (FR) characteristics. The formation and release of biofilm of the marine bacterium Cellulophaga lytica, the attachment and release of the microalga Navicula incerta, and the fraction removal and critical removal stress of reattached adult barnacles of Amphibalanus amphitrite were evaluated in laboratory assays. Correlations of AF/FR performance with surface characteristics such as wettability, surface energy, elastic modulus, and surface roughness were examined. Several of the xerogel coating compositions performed well against both microfouling organisms while the commercial coatings performed less well toward the removal of microalgae. Reattached barnacle adhesion as measured by critical removal stress was significantly lower on the commercial coatings when compared to the xerogel coatings. However, two xerogel compositions showed release of 89-100% of reattached barnacles. These two formulations were also tested in the field and showed similar results.     

Silicone fouling-release coatings: effects of the molecular weight of poly(dimethylsiloxane) and tetraethyl orthosilicate on the magnitude of pseudobarnacle adhesion strength

A series of poly(dimethyl siloxane) (PDMS)/silica nanocomposites were synthesized utilizing a sol gel method. The samples were evaluated using pseudobarnacle adhesion and tensile strength tests. The effects of the molecular weight of the PDMS and the size and structure of the silica domains on biofouling release and the mechanical behavior of the PDMS/silica materials were investigated. Three different molecular weights (18,000, 49,000 and 79,000 g mol(-1)) of hydroxyl-terminated PDMS (HT-PDMS) were used to prepare the nanocomposites with three different weight ratios (1:1, 3:1 and 5:1) of HT-PDMS to tetraethyl orthosilicate (TEOS). TEOS served as a crosslinker to form PDMS networks and as a precursor to form silica domains. Two different variants of TEOS with regard to its degree of polymerization (n) (monomeric type: n ≈= 1 and oligomeric type: n ≈= 5) were used for in situ formation of silica particles via the sol-gel process. The mechanical properties of the composites were characterized using stress-strain isotherms. All the mechanical properties evaluated (Young's modulus, tensile strength, energy required for rupture, elongation at break) improved with increases in the molecular weight of the HT-PDMS and the silica content. The pseudobarnacle adhesion test was used to examine the fouling- release (FR) properties of coatings applied on aluminum plates. The rupture energy and tensile strength increased substantially when oligomeric TEOS was employed in the PDMS/silica composites. Scanning electron microscopy (SEM) was used to investigate the structure of the silica domains. It was found that the use of oligomeric TEOS in higher molecular weight PDMS samples with higher PDMS/TEOS weight ratios led to low pseudobarnacle adhesion strengths of ≈ 0.3 MPa, which is in the range of commercial FR coatings.     

On-line monitoring of antifouling and fouling-release surfaces using bioluminescence and fluorescence measurements during laminar flow

A laminar flow biofilm-monitoring system was used to determine the efficacies of three antifouling (AF) coatings and five fouling-release (FR) coatings againstVibrio harveyi attachment. On-line measurements of tryptophan fluorescence and bioluminescence from each coating, normalized to an upstream stainless steel coupon, were used to determine the effects of AF and FR surfaces on biofilm formation. The AF coatings consisted of 5, 10, and 35 wt% Sea Nine 211 (C9211) incorporated into a vinyl copolymer. Both the 10 and 35 wt% coatings significantly inhibited biofilm biomass development measured by tryptophan fluorescence compared to the stainless steel control.V. harveyi bioluminescence was significantly greater than tryptophan fluorescence in cells attached to these coatings, suggesting that bioluminescence expression may be a marker for cellular stress or toxicity in biofilms. Five different polydimethylsiloxane (PDMS) FR coatings did not inhibit biofilm formation under low flow conditions. However, four PDMS coatings demonstrated decreased biomass levels compared to stainless steel after exposure to a shear stress of 330 dynes cm^–2. There was no toxic additive in these coatings; bioluminescence and tryptophan fluorescence were proportional.     

Rapid molecular methods for in‐field and laboratory identification of the yellow‐legged Asian hornet (Vespa velutina nigrithorax)

The yellow‐legged Asian hornet (Vespa velutina nigrithorax) is an invasive species that presents a threat to apiculture in Europe; first introduced into France in 2004, it has subsequently spread into neighbouring European countries. There is a risk of invasion and establishment in the UK, and in 2016, nests were found and destroyed in Alderney in the Channel Islands, and in Tetbury, Gloucestershire, illustrating a need for screening of suspect specimens so that invading hornets can be rapidly identified, and their nests destroyed. In this study, loop‐mediated isothermal amplification (LAMP) and real‐time PCR assays were developed to enable both in‐field and laboratory testing. Species‐specific identification assays and generic invertebrate control assays were developed. All the assays were validated according to the European Plant Protection Organisation standard PM 7/98. The assays were tested successfully against V. velutina nigrithorax obtained from France, Asia and the UK. Eight non‐target species, that were closely related or morphologically similar to the Asian hornet, gave negative results with the species‐specific assays, and positive results with the control assays. The assays could be used to detect target DNA at concentrations as low as 5 pg per reaction. LAMP was rapid, and cable of generating positive results within 10 min. Using simplified sample homogenization protocols that could be performed in the field, the LAMP assay was successful when tested against all developmental stages and nest samples, assisting with identification of samples that cannot be determined morphologically and allowing detection away from the laboratory. These assays provide a valuable tool for fast and reliable detection of this invasive species, offering the ability to identify damaged/incomplete specimens and immature life‐stages.     

Myocardial Dysfunction Induced by Food Restriction is Related to Morphological Damage in Normotensive Middle-Aged Rats

Summary Previous works from our laboratory have revealed that food restriction (FR) promotes discrete myocardial dysfunction in young rats. We examined the effects of FR on cardiac function, in vivo and in vitro, and ultrastructural changes in the heart of middle-aged rats. Twelve-month-old Wistar-Kyoto rats were fed a control (C) or restricted diet (daily intake reduced to 50% of the control group) for 90 days. Cardiac performance was studied by echocardiogram and in isolated left ventricular (LV) papillary muscle by isometric contraction in basal condition, after calcium chloride (5.2 mM) and beta-adrenergic stimulation with isoproterenol (10⁻⁶ M). FR did not change left ventricular function, but increased time to peak tension, and decreased maximum rate of papillary muscle tension development. Inotropic maneuvers promoted similar effects in both groups. Ultrastructural alterations were seen in most FR rat muscle fibers and included, absence and/or disorganization of myofilaments and Z line, hyper-contracted myofibrils, polymorphic and swollen mitochondria with disorganized cristae, and a great quantity of collagen fibrils. In conclusion, cardiac muscle sensitivity to isoproterenol and elevation of extracellular calcium concentration is preserved in middle-aged FR rats. The intrinsic muscle performance depression might be related to morphological damage.     

Poly(dimethyl siloxane) (PDMS) network blends of amphiphilic acrylic copolymers with poly(ethylene glycol)-fluoroalkyl side chains for fouling-release coatings. II. Laboratory assays and field immersion trials

Amphiphilic copolymers containing different amounts of poly(ethylene glycol)-fluoroalkyl acrylate and polysiloxane methacrylate units were blended with a poly(dimethyl siloxane) (PDMS) matrix in different proportions to investigate the effect of both copolymer composition and loading on the biological performance of the coatings. Laboratory bioassays revealed optimal compositions for the release of sporelings of Ulva linza, and the settlement of cypris larvae of Balanus amphitrite. The best-performing coatings were subjected to field immersion tests. Experimental coatings containing copolymer showed significantly reduced levels of hard fouling compared to the control coatings (PDMS without copolymer), their performance being equivalent to a coating based on Intersleek 700?. XPS analysis showed that only small amounts of fluorine at the coating surface were sufficient for good antifouling/fouling-release properties. AFM analyses of coatings under immersion showed that the presence of a regular surface structure with nanosized domains correlated with biological performance.     

A comparison between different fouling-release elastomer coatings containing surface-active polymers

Surface-active polymers derived from styrene monomers containing siloxane (S), fluoroalkyl (F) and/or ethoxylated (E) side chains were blended with an elastomer matrix, either poly(dimethyl siloxane) (PDMS) or poly(styrene-b-(ethylene-co-butylene)-b-styrene) (SEBS), and spray-coated on top of PDMS or SEBS preformed films. By contact angle and X-ray photoelectron spectroscopy measurements, it was found that the surface-active polymer preferentially populated the outermost layers of the coating, despite its low content in the blend. However, the self-segregation process and the response to the external environment strongly depended on both the chemistry of the polymer and the type of matrix used for the blend. Additionally, mechanical testing showed that the elastic modulus of SEBS-based coatings was one order of magnitude higher than that of the corresponding PDMS-based coatings. The coatings were subjected to laboratory bioassays with the marine alga Ulva linza. PDMS-based coatings had superior fouling-release properties compared to the SEBS-based coatings.     

Modulation of Hepatocarcinoma Cell Morphology and Activity by Parylene-C Coating on PDMS

Background

The ability to understand and locally control the morphogenesis of mammalian cells is a fundamental objective of cell and developmental biology as well as tissue engineering research. We present parylene-C (ParC) deposited on polydimethylsiloxane (PDMS) as a new substratum for in vitro advanced cell culture in the case of Human Hepatocarcinoma (HepG2) cells.

Principal Findings

Our findings establish that the intrinsic properties of ParC-coated PDMS (ParC/PDMS) influence and modulate initial extracellular matrix (ECM; here, type-I collagen) surface architecture, as compared to non-coated PDMS substratum. Morphological changes induced by the presence of ParC on PDMS were shown to directly affect liver cell metabolic activity and the expression of transmembrane receptors implicated in cell adhesion and cell-cell interaction. These changes were characterized by atomic force microscopy (AFM), which elucidated differences in HepG2 cell adhesion, spreading, and reorganization into two- or three-dimensional structures by neosynthesis of ECM components. Local modulation of cell aggregation was successfully performed using ParC/PDMS micropatterns constructed by simple microfabrication.

Conclusion/Significance

We demonstrated for the first time the modulation of HepG2 cells'' behavior in relation to the intrinsic physical properties of PDMS and ParC, enabling the local modulation of cell spreading in a 2D or 3D manner by simple microfabrication techniques. This work will provide promising insights into the development of cell-based platforms that have many applications in the field of in vitro liver tissue engineering, pharmacology and therapeutics.     

Fluorine-free mixed amphiphilic polymers based on PDMS and PEG side chains for fouling release applications

Fluorine-free mixed amphiphilic block copolymers with mixtures of short side groups of polydimethyl siloxane (PDMS) and polyethylene glycol (PEG) were synthesized and studied for their ability to influence the surface properties and control the adhesion of marine organisms to coated surfaces. The settlement (attachment) and strength of adhesion of two different marine algae, the green seaweed Ulva and the diatom Navicula, were evaluated against the surfaces. It is known that hydrophobic coatings based on polydimethyl siloxane elastomers (PDMSe) are prone to protein adsorption and accumulation of strongly adherent diatom slimes, in contrast to PEG-based hydrophilic surfaces that inhibit protein adsorption and moderate only weak adhesion of diatoms. By incorporating both PDMS and PEG side chains into the polymers, the effect of incorporating both polar and non-polar groups on fouling-release could be studied. The dry surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The ability of these mixed amphiphilic polymers to reconstruct in water was examined using underwater bubble contact angle and dynamic water contact angle experiments. To understand more about surface reconstruction behavior, protein adsorption experiments were carried out with fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) on both dry and pre-soaked surfaces.     

Assessment of laboratory methods for evaluating cassava genotypes for resistance to root rot disease

Field evaluation of six cassava genotypes for resistance to root rot disease was compared with three rapid laboratory methods (whole root inoculation, root slice inoculation, and stem inoculation) for resistance screening. Both the field evaluation and the three laboratory methods separated the varieties into resistant and susceptible groups. Genotypes 30572 and 91/02324 were resistant while 92/0247, 92/0057 and TME-1 were susceptible. One genotype (30001) was not consistent in its reaction between field evaluation and laboratory assays. In the laboratory assays with three fungal pathogens, different pathogens varied in their levels of virulence on host genotypes. With the most virulent pathogen (Botryodiplodia theobromae), the majority of the genotypes reacted in the same way across trials with the root slice and whole root assays. Due to the good correlation between the whole root assay and the field results, we recommend this for the routine assessment of cassava resistance to root rot disease and for the analysis of virulence of pathogen isolates. However, because of the advantages in terms of economy of labour, space, time, quantity of root and inoculum required, the root slice assay could be used for the preliminary screening of large cassava accessions. The selected genotypes can then be further screened with the whole root inoculation method.     

Two novel non-destructive biomarkers to assess PAH-induced oxidative stress and porphyrinogenic effects in crabs

Two novel, non-destructive assays were developed to evaluate contaminant-induced lipid peroxidation (thiobarbituric acid-reacting substances, TBARS, levels) and haem biosynthesis disruption (porphyrin excretion) in decapod crabs. A laboratory experiment was conducted whereby pie-crust crabs (Cancer novaezelandiae) were fed cockles (Austrovenus stutchburyi) collected from a contaminated and reference site and TBARS levels and porphyrin excretion determined using fluorometric analysis in urine samples. Pyrene metabolite levels were also measured in the same urine samples to assess polycyclic aromatic hydrocarbon (PAH) exposure. Contaminant-exposed crabs exhibited elevated urinary TBARS and porphyrin levels and a strong correlation was found between these two assays and the urinary pyrene metabolite concentrations. However, there was large within-treatment variability, which precluded a clear separation between the control and the impacted group. Nevertheless, consistency in the direction of the response shows that the biomarkers reflect pollutant levels and validates the use of these simple techniques from human medicine for environmental assessments.     

Amphiphilic block copolymer/poly(dimethylsiloxane) (PDMS) blends and nanocomposites for improved fouling-release

Amphiphilic diblock copolymers, Sz6 and Sz12, consisting of a poly(dimethylsiloxane) block (average degree of polymerisation = 132) and a PEGylated-fluoroalkyl modified polystyrene block (Sz, average degree of polymerisation = 6, 12) were prepared by atom transfer radical polymerization (ATRP). Coatings were obtained from blends of either block copolymer (1–10 wt%) with a poly(dimethylsiloxane) (PDMS) matrix. The coating surface presented a simultaneous hydrophobic and lipophobic character, owing to the strong surface segregation of the lowest surface energy fluoroalkyl chains of the block copolymer. Surface chemical composition and wettability of the films were affected by exposure to water. Block copolymer Sz6 was also blended with PDMS and a 0.1 wt% amount of multiwall carbon nanotubes (CNT). The excellent fouling-release (FR) properties of these new coatings against the macroalga Ulva linza essentially resulted from the inclusion of the amphiphilic block copolymer, while the addition of CNT did not appear to improve the FR properties.     

Fluorinated/siloxane copolymer blends for fouling release: chemical characterisation and biological evaluation with algae and barnacles

Fouling-release coatings were prepared from blends of a fluorinated/siloxane copolymer with a poly(dimethyl siloxane) (PDMS) matrix in order to couple the low modulus character of PDMS with the low surface tension typical for fluorinated polymers. The content of the surface-active copolymer was varied in the blend over a broad range (0.15–10 wt % with respect to PDMS). X-ray photoelectron spectroscopy depth profiling analyses were performed on the coatings to establish the distribution of specific chemical constituents throughout the coatings, and proved enrichment in fluorine of the outermost layers of the coating surface. Addition of the fluorinated/siloxane copolymer to the PDMS matrix resulted in a concentration-dependent decrease in settlement of barnacle, Balanus amphitrite, cyprids. The release of young plants of Ulva, a soft fouling species, and young barnacles showed that adhesion strength on the fluorinated/siloxane copolymer was significantly lower than the siloxane control. However, differences in adhesion strength were not directly correlated with the concentration of copolymer in the blends.     

Microfluidic accumulation assay probes attachment of biofilm forming diatom cells

Testing of fouling release (FR) technologies is of great relevance for discovery of the next generation of protective marine coatings. In this paper, an accumulation assay to test diatom interaction under laminar flow with the model organism Navicula perminuta is introduced. Using time lapse microscopy with large area sampling allows determination of the accumulation kinetics of the diatom on three model surfaces with different surface properties at different wall shear stresses. The hydrodynamic conditions within the flow cell are described and a suitable shear stress range to perform accumulation experiments is identified at which statistically significant discrimination of surfaces is possible. The observed trends compare well to published adhesion preferences of N. perminuta. Also, previously determined trends of critical wall shear stresses required for cell removal from the same set of functionalized interfaces shows consistent trends. Initial attachment mediated by extracellular polymeric substances (EPS) present outside the diatoms leads to the conclusion that the FR potential of the tested coating candidates can be deducted from dynamic accumulation experiments under well-defined hydrodynamic conditions. As well as testing new coating candidates for their FR properties, monitoring of the adhesion process under flow provides additional information on the mechanism and geometry of attachment and the population kinetics.     

Amphiphilic hydrolyzable polydimethylsiloxane-b-poly(ethyleneglycol methacrylate-co-trialkylsilyl methacrylate) block copolymers for marine coatings. II. Antifouling laboratory tests and field trials

Abstract

Poly(dimethylsiloxane) (PDMS) elastomer coatings containing an amphiphilic hydrolyzable diblock copolymer additive were prepared and their potential as marine antifouling and antiadhesion materials was tested. The block copolymer additive consisted of a PDMS first block and a random poly(trialkylsilyl methacrylate (TRSiMA, R?=?butyl, isopropyl)-co-poly(ethyleneglycol) methacrylate (PEGMA) copolymer second block. PDMS-b-TRSiMA block copolymer additives without PEGMA units were also used as additives. The amphiphilic character of the coating surface was assessed in water using the captive air bubble technique for measurements of static and dynamic contact angles. The attachment of macro- and microorganisms on the coatings was evaluated by field tests and by performing adhesion tests to the barnacle Amphibalanus amphitrite and the green alga Ulva rigida. All the additive-based PDMS coatings showed better antiadhesion properties to A. amphitrite larvae than to U. rigida spores. Field tests provided meaningful information on the antifouling and fouling release activity of coatings over an immersion period of 23?months.